| Autor: |
Hee Jun Ahn, Hyungjin Kim, Dong Hoon Jin, Nam Joo Hong |
| Rok vydání: |
2015 |
| Předmět: |
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| Zdroj: |
Bulletin of the Korean Chemical Society. 36:1885-1896 |
| ISSN: |
1229-5949 |
| DOI: |
10.1002/bkcs.10367 |
| Popis: |
A new and relatively simple spectrophotometric technique has been developed for the accurate determination of α-factor pheromone affinities for Ste2p whole cell receptor in yeast a-cells. We designed and tested nine detector peptides containing mono- (e412 = 14 500) or tri-cysteine residues (e412 = 43 660). The free unbound detector was detected using Ellman's reagent at 412 nm. Saturation binding studies using Saccharomyces cerevisiae Y 7925 ( MATa ) at a concentration of 2.5 × 1011 cells/mL with the highest affinity detector 1, [Orn6]α-factor-[Cys]3, resulted in a dissociation constant ( K D ) of 1.67 × 10−7 and total binding sites per cell (B cell = 29 500 sites/cell) comparable with those obtained using radiolabeled binding assays. Competitive binding assay using five nonchromogenic α-factor analogs allowed for the determination of each K D value. [Orn6,d-Ala9]α-factor showed the highest receptor affinity ( K D = 1.03 × 10−7 M), which was threefold higher than that of native α-factor. This assay provides rapid and convenient results for determining the relative affinities of nonchromogenic α-factors and eliminates the need for radioactive waste disposal. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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